The fields of molecular electronics/photonics and nanotechnology offer immense technological promise for the future. Nanotechnology is defined as a projected technology based on a generalized ability to build objects to complex atomic specifications. Drexler, Proc. Natl. Acad. Sci USA, 78:5275-5278, (1981). Nanotechnology means an atom-by-atom or molecule-by-molecule control for organizing and building complex structures all the way to the macroscopic level. Nanotechnology is a bottom-up approach, in contrast to a top-down strategy like present lithographic techniques used in the semiconductor and integrated circuit industries. The success of nanotechnology will be based on the development of programmable self-assembling molecular units and molecular level machine tools, so-called assemblers, which will enable the construction of a wide range of molecular structures and devices. Drexler, in “Engines of Creation,” Doubleday Publishing Co., New York, N.Y. (1986). Thus, one of the first and most important goals in nanotechnology is the development of programmable self-assembling molecular construction units.
Present molecular electronic/photonic technology includes numerous efforts from diverse fields of scientists and engineers. Carter, ed. in “Molecular Electronic Devices II,” Marcel Dekker, Inc, New York, N.Y. (1987). Those fields include organic polymer based rectifiers, Metzger et al., in “Molecular Electronic Devices II,” Carter, ed., Marcel Dekker, New York, N.Y., pp. 5-25 (1987), conducting conjugated polymers, MacDiarmid et al., Synthetic Metals, 18:285 (1987), electronic properties of organic thin films or Langmuir-Blogett films, Watanabe et al., Synthetic Metals, 28:C473 (1989), molecular shift registers based on electron transfer, Hopfield et al., Science, 241:817 (1988), and a self-assembly system based on synthetically modified lipids which form a variety of different “tubular” microstructures. Singh et al., in “Applied Bioactive Polymeric Materials,” Plenum Press, New York, N.Y., pp. 239-249 (1988). Molecular optical or photonic devices based on conjugated organic polymers, Baker et al., Synthetic Metals, 28:D639 (1989), and nonlinear organic materials have also been described. Potember et al., Proc. Annual Conf. IEEE in Medicine and Biology, Part 4/6:1302-1303 (1989).
However, none of the cited references describe a sophisticated or programmable level of self-organization or self-assembly. Typically the actual molecular component which carries out the electronic and/or photonic mechanism is a natural biological protein or other molecule. Akaike et al., Proc. Annual Conf. IEEE in Medicine and Biology, Part 4/6:1337-1338 (1989). There are presently no examples of a totally synthetic programmable self-assembling molecule which produces an efficient electronic or photonic structure, mechanism or device.
Progress in understanding self-assembly in biological systems is relevant to nanotechnology. Drexler, Proc. Natl. Acad. Sci USA, 78:5275-5278 (1981). Drexler, in “Engines of Creation,” Doubleday Publishing Co., New York, N.Y. (1986). Areas of significant progress include the organization of the light harvesting photosynthetic systems, the energy transducing electron transport systems, the visual process, nerve conduction and the structure and function of the protein components which make up these systems. The so called bio-chips described the use of synthetically or biologically modified proteins to construct molecular electronic devices. Haddon et al., Proc. Natl. Acad. Sci. USA, 82:1874-1878 (1985). (McAlear et al., in “Molecular Electronic Devices II,” Carter ed., Marcel Dekker, Inc., New York N.Y., pp. 623-633 (1987). Some work on synthetic proteins (polypeptides) has been carried out with the objective of developing conducting networks. McAlear et al., in “Molecular Electronic Devices,” Carter ed., Marcel Dekker, New York, N.Y., pp. 175-180 (1982). Other workers have speculated that nucleic acid based bio-chips may be more promising. Robinson et al., “The Design of a Biochip: a Self-Assembling Molecular-Scale Memory Device,” Protein Engineering, 1:295-300 (1987).
Great strides have also been made in our understanding of the structure and function of the nucleic acids, deoxyribonucleic acid or DNA, Watson, et al., in “Molecular Biology of the Gene,” Vol. 1, Benjamin Publishing Co., Menlo Park, Calif. (1987), which is the carrier of genetic information in all living organisms. In DNA, information is encoded in the linear sequence of nucleotides by their base units adenine, guanine, cytosine, and thymidine (A, G, C, and T). Single strands of DNA (or polynucleotides) have the unique property of recognizing and binding, by hybridization, to their complementary sequence to form a double stranded nucleic acid duplex structure. This is possible because of the inherent base-pairing properties of the nucleic acids; A recognizes T, and G recognizes C. This property leads to a very high degree of specificity since any given polynucleotide sequence will hybridize only to its exact complementary sequence.
In addition to the molecular biology of nucleic acids, great progress has also been made in the area of the chemical synthesis of nucleic acids (16). This technology has developed so automated instruments can now efficiently synthesize sequences over 100 nucleotides in length, at synthesis rates of 15 nucleotides per hour. Also, many techniques have been developed for the modification of nucleic acids with functional groups, including: fluorophores, chromophores, affinity labels, metal chelates, chemically reactive groups and enzymes. Smith et al., Nature, 321:674-679 (1986); Agarawal et al., Nucleic Acids Research, 14:6227-6245 (1986); Chu et al., Nucleic Acids Research, 16:3671-3691 (1988).
An impetus for developing both the synthesis and modification of nucleic acids has been the potential for their use in clinical diagnostic assays, an area also referred to as DNA probe diagnostics. Simple photonic mechanisms have been incorporated into modified oligonucleotides in an effort to impart sensitive fluorescent detection properties into the DNA probe diagnostic assay systems. This approach involved fluorophore and chemiluminescent-labeled oligonucleotides which carry out Förster nonradiative energy transfer. Heller et al., in “Rapid Detection and Identification of Infectious Agents,” Kingsbury et al., eds., Academic Press, New York, N.Y. pp. 345-356 (1985). Förster nonradiative energy transfer is a process by which a fluorescent donor (D) group excited at one wavelength transfers its absorbed energy by a resonant dipole coupling process to a suitable fluorescent acceptor (A) group. The efficiency of energy transfer between a suitable donor and acceptor group has a 1/r6 distance dependency (see Lakowicz et al., in “Principles of Fluorescent Spectroscopy,” Plenum Press, New York, N.Y., Chap. 10, pp. 305-337 (1983)).
In the work of Heller et al., supra, two fluorophore labeled oligonucleotides are designed to bind or hybridize to adjacent positions of a complementary target nucleic acid strand and then produce efficient fluorescent energy transfer in terms of re-emission by the acceptor. The first oligonucleotide is labeled in the 3′ terminal position with a suitable donor group, and the second is labeled in the 5′ terminal position with a suitable acceptor group. The binding or hybridization to the complementary sequence positions the fluorescent donor group and fluorescent acceptor groups so they are at optimal distance (theoretically) for most efficient Förster nonradiative energy transfer. However, the observed energy transfer efficiency in terms of re-emission by the acceptor was relatively low (˜20%) for this particular arrangement.
In later work (Heller et al., European Patent Application No. EPO 0229943, 1987; and Heller et al., U.S. Pat. No. 4,996,143, Feb. 26, 1991), the advances in synthetic chemistry provided methods for the attachment of fluorophores at any position within an oligonucleotide sequence using a linker arm modified nucleotide. Also, with this synthetic linkage technique it was possible to incorporate both a donor and an acceptor fluorophore within the same oligonucleotide. Using the particular linker arm, it was found that the most efficient energy transfer (in terms of re-emission by the acceptor) occurred when the donor and acceptor were spaced by 5 intervening nucleotide units, or approximately 1.7 nm apart. Heller et al., U.S. Pat. No. 4,996,143 also showed that as the nucleotide spacing decreases from 4 to 0 units (1.4 nm to 0 nm), the energy transfer efficiency also decreases; which is not in accordance with Förster theory. As the nucleotide spacing was increased from 6 to 12 units (2 nm to 4.1 nm), the energy transfer efficiency was also found to decrease; which is in accordance with Förster theory. At the time, it was not explained nor understood why the more closely spaced donor and acceptor arrangements had reduced energy transfer efficiency and were not in agreement with Förster theory. In particular, the teachings of Heller et al. did not address multiple donor resonance and extended energy transfer from donors beyond Förster distances of >5 nm.
Fluorescent energy transfer has been utilized in other areas which include immunodiagnostics and liquid chromatography analysis. Morrison et al., Anal. Biochem., 174:101-120 (1988); and Garner et al., Anal. Chem., 62:2193-2198 (1990). Also, some of the initial demonstrations of simple fluorescent donor/acceptor energy transfer in nucleic acids were later corroborated by other workers. Cardullo et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988); and Morrision et al., Anal. Biochem., 183:231-244 (1989). In the Cardullo et al. work, an arrangement is studied where two short (12-mer) oligonucleotide sequences, each terminally labeled with rhodamine acceptors and hybridized to a complementary 29-mer sequence, are associated with several intercalating donors (acridine orange). The arrangements described by Cardullo show some added energy transfer due to the additional donors. However, this increase in energy transfer efficiency is entirely consistent with direct donor to acceptor transfer, as none of the donors were described as functioning beyond the Förster distance necessary for efficient transfer. To date, there has been no descriptions of an organized structure capable of extended energy transfer from multiple donors and to an acceptor beyond normal Förster distances.